RESUMO
Abstract The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.
Resumo O objetivo deste estudo foi avaliar a expressão de MMP2 e MMP9 durante a progressão da periodontite apical (AP) em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO) comparados aos camundongos wild type (WT). A AP foi induzida nos primeiros molares inferiores dos camundongos TLR2 KO (n = 18), MyD88 KO (n = 18) e WT (n = 18). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. As lâminas foram coradas por imuno-histoquímica e analisadas para a detecção de MMP2 e MMP9. A análise estatística semi-quantitativa da imuno-histoquímica foi realizada pelo teste qui-quadrado (α = 0,05). Nos períodos iniciais de progressão AP, foi observada uma expressão aumentada de MMP9 nos camundongos TLR2 KO e MyD88 KO. Nos períodos finais de progressão AP, observou-se uma redução da expressão de MMP2 e um aumento da expressão de MMP9 nos camundongos TLR2 KO. A produção de MMP2 e MMP9 foi modulada por TLR2 e MyD88 durante a progressão da periodontite apical.
Assuntos
Animais , Camundongos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fator 88 de Diferenciação Mieloide/fisiologia , Periodontite Periapical/enzimologia , Receptor 2 Toll-Like/fisiologia , Progressão da Doença , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Periodontite Periapical/metabolismo , Periodontite Periapical/patologiaRESUMO
The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.
Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fator 88 de Diferenciação Mieloide/fisiologia , Periodontite Periapical/enzimologia , Receptor 2 Toll-Like/fisiologia , Animais , Progressão da Doença , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Periodontite Periapical/metabolismo , Periodontite Periapical/patologiaRESUMO
AIM: To characterize the formation and progression of experimentally induced periapical lesions in teeth of MyD88 knockout (MyD88 KO) mice compared with wild-type (WT) mice. METHODOLOGY: Periapical lesions were induced in the mandibular first molars of 30 WT and 30 MyD88 KO mice. After 7, 21 and 42 days, the animals were euthanized and the mandibles were subjected to histotechnical processing. Histological sections were stained with haematoxylin and eosin (HE), TRAP histoenzymology, Brown and Brenn staining and immunohistochemistry (RANK, RANKL, OPG). Data were subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests and the Dunn post-test, using the SPSS software, version 17.0 (α = 0.05). RESULTS: Regarding the periapical lesion size, the MyD88 KO group had significantly higher values than the WT group in the periods of 7 (P = 0.001) and 21 days (P = 0.05). A larger number of neutrophils in the MyD88 KO group were observed (P = 0.01 at 7 days, P = 0.004 at 21 days and P < 0.001 at 42 days). Regarding the number of osteoclasts, no statistically significant difference was observed between the groups at any of the experimental periods (P = 0.884 at 7 days, P = 0.506 at 21 days and P = 0.211 at 42 days). CONCLUSIONS: In the absence of MyD88, the animals had larger periapical lesions, with a severe inflammatory infiltrate and a significantly larger number of neutrophils.
Assuntos
Fator 88 de Diferenciação Mieloide/fisiologia , Neutrófilos/patologia , Periodontite Periapical/fisiopatologia , Animais , Cavidade Pulpar/patologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220(pos). TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220(pos) (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220(neg) in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220(pos) (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220(pos) (MyD88-independent) and ASC B220(neg) into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220(neg) in peritoneum and BM. Terminally differentiated ASC B220(neg) required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220(pos) rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220(pos) and ASC B220(neg).
Assuntos
Células Produtoras de Anticorpos/fisiologia , Diferenciação Celular/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Most snake accidents in North Brazil are attributed to Bothrops atrox, a snake species of the Viperidae family whose venom simultaneously induces local and systemic effects in the victims. The former are clinically more important than the latter, as they cause severe tissue lesions associated with strong inflammatory responses. Although several studies have shown that inflammatory mediators are produced in response to B. atrox venom (BaV), there is little information concerning the molecular pathways involved in innate immune system signaling. Myeloid differentiation factor 88 (MyD88) is an adaptor molecule responsible for transmitting intracellular signals from most toll-like receptors (TLRs) after they interact with pathogen-associated molecular patterns (PAMPs) or other stimuli such as endogenous damage-associated molecular patterns (DAMPs). The MyD88-dependent pathway leads to activation of transcription factors, which in turn induce synthesis of inflammatory mediators such as eicosanoids, cytokines and chemokines. The aim of this study was to investigate the involvement of MyD88 on the acute inflammatory response induced by BaV. Wild-type (WT) C57BL/6 mice and MyD88 knockout (MyD88(-/-)) mice were intraperitoneally injected with BaV. Compared to WT mice, MyD88(-/-) animals showed an impaired inflammatory response to BaV, with lower influx of polymorphonuclear and mononuclear cells to the peritoneal cavity. Furthermore, peritoneal leukocytes from BaV-injected MyD88(-/-) mice did not induce COX-2 or LTB4 protein expression and released low concentrations of PGE2. These mice also failed to produce Th1 and Th17 cytokines and CCL-2, but IL-10 levels were similar to those of BaV-injected WT mice. Our results indicate that MyD88 signaling is required for activation of the inflammatory response elicited by BaV, raising the possibility of developing new therapeutic targets to treat Bothrops sp. poisoning.
Assuntos
Bothrops/fisiologia , Venenos de Crotalídeos/toxicidade , Inflamação/induzido quimicamente , Fator 88 de Diferenciação Mieloide/fisiologia , Animais , Citocinas/metabolismo , Dinoprostona/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Brucella ovis is an important cause of epididymitis in rams, which results in impaired fertility and economic losses. This study demonstrated the role of TLR during the acute phase of infection in the mouse model. C57BL/6 wild type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice were infected with B. ovis and bacteriology, histopathology, and pro-inflammatory gene expression were evaluated at 7days post-infection. MyD88(-/-) mice had higher bacterial loads in the spleen when compared to wild type mice. This enhanced susceptibility was associated with decreased inflammatory response in the liver. TLR9(-/-) mice also had higher bacterial loads when compared to wild type mice, but, surprisingly, they developed stronger inflammatory response. TLR2(-/-) and TLR4(-/-) mice were as susceptible as wild type mice to B. ovis infection. Therefore, MyD88 and TLR9 are required for controlling B. ovis multiplication during the early stages of infection.
Assuntos
Brucelose/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Receptor Toll-Like 9/fisiologia , Animais , Carga Bacteriana/veterinária , Brucella ovis , Brucelose/imunologia , Suscetibilidade a Doenças/veterinária , Feminino , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Fator 88 de Diferenciação Mieloide/genética , Baço/microbiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/fisiologia , Receptor Toll-Like 9/genéticaRESUMO
Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. In this manuscript, we described an immune mechanism for inflammatory bone loss in response to infection by Brucella abortus. We established a requirement for MyD88 and TLR2 in TNF-α-elicited osteoclastogenesis in response to B. abortus infection. CS from macrophages infected with B. abortus induced BMM to undergo osteoclastogenesis. Although B. abortus-infected macrophages actively secreted IL-1ß, IL-6, and TNF-α, osteoclastogenesis depended on TNF-α, as CS from B. abortus-infected macrophages failed to induce osteoclastogenesis in BMM from TNFRp55â»/â» mice. CS from B. abortus-stimulated MyD88â»/â» and TLR2â»/â» macrophages failed to express TNF-α, and these CS induced no osteoclast formation compared with that of the WT or TLR4â»/â» macrophages. Omp19, a B. abortus lipoprotein model, recapitulated the cytokine production and subsequent osteoclastogenesis induced by the whole bacterium. All phenomena were corroborated using human monocytes, indicating that this mechanism could play a role in human osteoarticular brucellosis. Our results indicate that B. abortus, through its lipoproteins, may be involved in bone resorption through the pathological induction of osteoclastogenesis.
Assuntos
Brucella abortus/fisiologia , Macrófagos Peritoneais/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Osteoclastos/fisiologia , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Antígenos de Bactérias/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Brucelose/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Feminino , Humanos , Lipoproteínas/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Ligante RANK/biossíntese , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/deficiência , Fator de Crescimento Transformador beta/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The role of innate cells and their receptors within the male genital tract remains poorly understood. Much less is known about the relative contribution of different genital tract cells such as epithelial/stromal cells and resident leucocytes. In this study, we examined innate immune responses to Chlamydia trachomatis by prostate epithelial/stromal cells and prostate resident leucocytes. Murine prostate primary cultures were performed and leucocyte and epithelial/stromal cells were sorted based on surface protein expression of CD45 by magnetism-activated cell sorting or fluorescence-activated cell sorting. Prostate derived CD45- and CD45+ cells were infected with C. trachomatis and chemokine secretion assayed by ELISA. Similar experiments were performed using prostate CD45+ and CD45- cells from myeloid differentiation factor 88 (Myd88(-/-)) mice or toll-like receptor (Tlr2(-/-)) and Tlr4(mutant) double-deficient mice. Moreover, a TLR-signalling pathway array was used to screen changes in different genes involved in TLR-signalling pathways by real-time PCR. Prostate derived CD45- and CD45+ cells responded to chlamydial infection with the production of different chemokines. Both populations expressed genes involved in TLR signalling and required to respond to pathogen-associated molecular patterns and to C. trachomatis infection. Both populations required the adaptor molecule MYD88 to elicit chemokine response against C. trachomatis. TLR2-TLR4 was essential for chemokine production by CD45+ prostate derived cells, but in their absence, CD45- cells still produced significant levels of chemokines. We demonstrate that C. trachomatis is differentially recognised by prostate derived CD45+ and CD45- cells and suggest that diverse strategies are taking place in the local microenvironment of the host in response to the infection.
Assuntos
Quimiocinas/metabolismo , Infecções por Chlamydia/patologia , Chlamydia trachomatis/fisiologia , Antígenos Comuns de Leucócito/metabolismo , Próstata/metabolismo , Próstata/patologia , Animais , Células Cultivadas , Quimiocina CXCL1/metabolismo , Infecções por Chlamydia/genética , Infecções por Chlamydia/metabolismo , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Cultura Primária de Células , Próstata/microbiologia , Regulação para CimaRESUMO
The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.
Assuntos
Fator 88 de Diferenciação Mieloide/fisiologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Imunidade Adaptativa/imunologia , Animais , Imunidade Inata/imunologia , Interleucina-18/biossíntese , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/fisiologiaRESUMO
Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-beta production and the expansion of CD4(+)CD25(+)Foxp3(+) cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.
Assuntos
Células Dendríticas/imunologia , Fasciola hepatica/imunologia , Proteínas de Helminto/fisiologia , Tolerância Imunológica , Células Mieloides/imunologia , Animais , Citocinas/biossíntese , Feminino , Fatores de Transcrição Forkhead/análise , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Células Th2/imunologia , Receptores Toll-Like/fisiologiaRESUMO
An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappaB-dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.
Assuntos
Doença de Chagas/imunologia , Imunidade Inata , Proteína Adaptadora de Sinalização NOD1/fisiologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/genética , Doença de Chagas/metabolismo , Predisposição Genética para Doença , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/fisiologia , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/fisiologiaRESUMO
Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.